Microbial fermentation is the method applied most widely to produce L-amino acid(s). The bacteria performance of producing amino acid(s) through fermentation is the key factor affecting whether the fermentation method can be applied at a large scale of industrialization. At present, there are still a few amino acids that are not realized to produce through the fermentation method due to absence of the production bacterial strain(s) with good fermentation performance. Moreover, as for the bacterial strains producing amino acids which have been realized to produce through the fermentation method, the amino acid tilter and yield from glucose still need to be improved in order to save production cost. For example, L-histidine is the ninth amino acid necessary to human and animal life. It plays the role in the fundamental physiological processes such as body growth and development, oxidation resistance and immuno-regulation and is an important amino acid for medical purpose. It can be used in infusion preparations for heart disease, anemia and gastrointestinal ulcer. Now L-histidine is mainly produced through the method of protein hydrolysis and extraction with pigs (cow) blood powder as the raw material. However, this method has the drawbacks such as high cost, low utilization of raw materials, complex extraction process and serious environmental pollution resulting in high production cost and high price. Nevertheless, the method of producing L-histidine through microbiological fermentation hasnot been applied at a large scale of industrialization. The bio-synthesis of L-histidine is featured in competing the precursor substances with nucleotide synthesis, complex metabolic regulation mechanism and high energy demand during synthesis process. Thus, the L-histidine production and yield of engineering bacteria are relatively low. The bacterial strains producing L-histidine are mainly bred through the methods of several rounds of traditional mutation-screening and genetic engineering on the basis of mutant strains. However, the strains produced from mutation-screening can accumulate a large amount of negative-effect mutation, resulting in slow growth of strains, reduced environmental tolerance and increased nutrients demand. Therefore, these drawbacks limit the industrial application of strains. Till now, there is only one report about the study of modifying and constructing L-histidine engineering bacteria through systems metabolic engineering (Doroshenko, V. G., Lobanov, A. O., Fedorina, E. A., 2013. The directed modification of Escherichia coli MG1655 to obtain histidine-producing mutants, Appl Biochem Microbiol. 49, 130-135.). This study uses the wild type of E. coli MG1655 as the starting bacteria and introduced E271K mutation into the gene hisG to weaken the feedback inhibitory regulation of histidine; knocks out the transcription attenuator hisL of the synthetic operon of histidine and enhanced the expression of the synthetic operon of histidine; also knocks out the gene purR and increased the synthesis of histidine synthetic precursor PRPP to construct a strain of engineering bacteria producing L-histidine. This study only modifies the terminal synthetic pathway of L-histidine and the yield of L-histidine is only 4.9 g/L. Thus, it is far from the realization of industrial application.
The L-histidine bio-synthesis is derived from the pentose phosphate pathway. When the glucose is used as the carbon source, the precursor for L-histidine synthesis-phosphoribosyl pyrophosphate (PRPP) is produced from the pentose phosphate pathway and PRPP simultaneously enters the synthetic pathway of nucleotide and the synthetic pathway of L-histidine where the former produces another precursor ATP for L-histidine synthesis.
In addition, the pentose phosphate pathway is also the main path forming cofactor(s) NADPH necessary to the synthesis of many amino acids (such as L-lysine, L-valine, L-threonine, L-proline, L-hydroxyproline), wherein: 4 molecules of NADPH are consumed to synthesize one molecule of L-lysine, 3 molecules of NADPH for 1 molecule of L-threonine, L-proline or L-hydroxyproline, and 2 molecules of NADPH for 1 molecule of L-valine.
The glucose-6-phosphate isomerase of the glycolytic pathway can be inactivated to guide the carbon metabolic flow to the pentose phosphate pathway. However, it will result in weakened growth of bacterial strains and glucose metabolism ability; hence, it is unfavorable for the application of bacterial strains in fermentation production (Marx, A., Hans, S., Mockel, B., Bathe, B., de Graaf, A. A., McCormack, A. C., Stapleton, C., Burke, K., O'Donohue, M., Dunican, L. K., 2003. Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum. J Biotechnol. 104, 185-197.). The results of previous studies by the inventor verified that: knocking out the encoding gene pgi of the glucose-6-phosphate isomerase resulted in serious degradation of strain growth and glucose metabolic ability and also decrease of the yield of L-histidine accordingly. Moreover, the inventor also found that it was not effective to improve the yield of L-histidine only through improving the expression of the glucose-6-phosphate dehydrogenase.